About The Example Experiments - abi 7500 Getting Started Manual

Chapter 1 Get Started

About the Example Experiments

About the Example Experiments

About the
Relative Standard
Curve Example
Experiment
Notes
14
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
The objective of the relative standard curve example experiment is to compare the
expression levels of the c-myc transcriptional factor (an oncoprotein that activates the
transcription of growth-associated genes) in liver and kidney tissues.
In the relative standard curve example experiment:
• The samples are cDNA prepared from total RNA isolated from liver and kidney tissues.
• The target is human c-myc.
• The endogenous control is human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
• The reference sample is RNA isolated from kidney tissue.
• One standard curve is set up for c-myc (target). The standard used for the standard
dilution series is a cDNA sample of known quantity prepared from RNA isolated from
lung tissue.
• One standard curve is set up for GAPDH (endogenous control). The standard used for the
standard dilution series is a cDNA sample of known quantity prepared from RNA isolated
from lung tissue.
• The experiment is designed for singleplex PCR, where the target (c-myc) and endogenous
control (GAPDH) assays are performed in separate wells.
• Reactions are set up for 2-step RT-PCR. The High-Capacity cDNA Reverse Transcription
Kit is used for reverse transcription; the TaqMan
PCR.
• Primer/probe sets are selected from the Applied Biosystems TaqMan
Assays product line:
– For the target assay (c-myc), the assay ID is Hs00153408_m1 (RefSeq NM_002467.3).
– For the endogenous control assay (GAPDH), the assay ID is Hs99999905_m1
(RefSeq NM_002046.3).
Reaction Plate Layout
The 7500 software displays the 96-well plate layout, as shown below.
®
Universal PCR Master Mix is used for
®
Gene Expression
and Comparative C Experiments
T